The present invention concerns an analysis method and a system for performing this analysis. Specifically the invention concerns a method for determination of hemoglobin in unaltered whole blood and a system which can be used in this determination.
A disposable cuvette for sampling a fluid, mixing the sample with a reagent and directly making optical analyses of the sample mixed with the reagent is previously known from U.S. Pat. No. 4,088,448. This known cuvette has several advantages as it i.a. simplifies the sampling procedure, reduces the number of utensils and considerably improves the accuracy of analysis by making the analysing procedure independent of the operating technique of the operator making the analysis. A cuvette construction based on the same principle and with improved flow characteristics is disclosed in the U.S. Pat. No. 5,674,457.
A disposable cuvette developed according to these patents is currently widely used for hemoglobin measurement (Hb determination) of undiluted whole blood. To this end the cuvette cavity has been pre-treated with a reagent, such that when a blood sample is drawn into the cuvette, the walls of the red blood cells are disintegrated and a chemical reaction is initiated. The result of the reaction allows Hb determination by absorption measurement directly through the transparent walls of the cuvette which, in the measuring zone, also called the optical window, has a predetermined and accurately defined distance between the inner surfaces of the opposing planar walls. The measurement method is based on a modified azidmethemoglobin method according to Vanzetti, G., Am.J. Lab. and Clin. Med. 67, 116 (1966).
The spectrophotometric measurements are made at 570 and 880 nm. This quantitative measurement method based on dry chemistry has met with considerable success as can be seen in e.g. the article by von Schenck et al in Clinical Chemistry, vol 32, No 3, 1986 as the method gives equal or even superior results in comparison with the results obtained with standardised wet methods for the determination of Hb. The reagent used is comprised of sodium deoxycholate which hemolyses the red blood cells, sodium azide and sodium nitrite, which converts hemoglobin to azidmethemoglobin.
Due to the hygroscopic properties of the reagents used, the shelf life is limited and the storage of the cuvettes in sealed packages including a drying agent is required. Even more troublesome is the fact that, in climates with high humidity, the cuvette has to be used within a few minutes after the removal from the package, as otherwise the reagents will be destroyed and the measurement will be inaccurate and thus useless.
The problems originating from the hygroscopic properties of the reagents used may however be eliminated as it has been found that these reagents must not be used as disclosed in the co-pending patent application PCT SE01/01442 according to which the first absorption measurement is performed at a wavelength range 490-520 nm directly on the sample in the microcuvette. According to the invention disclosed in this patent application it is however necessary that the blood is hemolysed before the measurement is performed. The cuvette cavity must thus include a hemolysing agent for disintegrating the red blood cells and releasing the hemoglobin contained in these cells. The necessity of using a hemolysing agent when performing photometric absorbance measurements of hemoglobin in a blood sample is also disclosed in e.g. the U.S. Pat. No. 5,064,282 (Artel).
Quantitative methods for optical determination of hemoglobin in whole blood without using hemolysing agent are known but these methods have in common that they are all comparatively complicated. This depends above all on the inhomogeneity of the blood due to the high concentration of red blood cells, a consequence of which is that light is scattered upon interaction with these particles of inhomogeneous blood samples. Accordingly the light is not transmitted directly through the sample but deflected over a range of scattering angles. Another factor that causes problems is the fact that blood may contain as many as five different species of hemoglobin. Patent publications addressing these problems are i.a. the U.S. Pat. No. 6,262,798 (Shepherd) and WO 01/53806 (Radiometer).
According to the invention disclosed in the U.S. Pat. No. 6,262,798 a plurality of wavelengths are needed in order to achieve a correct measurement. The fact that many wavelengths are needed makes the spectrophotometer comparatively complicated. The wavelengths are selected by their ability to distinguish the hemoglobin species at minimum scatter and maximum absorbance. The patent also discloses the use of a large detector which reduces the problem of scattering beyond the detection range.
WO 01/53806 discloses an apparatus which is especially applicable for optical measurements on whole blood. This apparatus comprises an absorption filter or an interference filter, which provides correction for variations in the detector sensitivity and in the effective optical path length as observed upon varying level of scattering. The apparatus uses a large detector for detecting scattered light transmitted through the absorption filter or the interference filter.
The finding according to the present invention that an accurate determination of the total amount of hemoglobin in whole blood can be made not only without using a hemolysing agent but also without using a plurality of wavelengths as disclosed in the U.S. Pat. No. 6,262,798 or a special absorption or interference filter which provides correction for variations in the detector sensitivity and in the effective optical path length as observed upon varying level of scattering as disclosed in WO 01/53806 was therefore most unexpected.
An object of the present invention is to provide a rapid, quantitative method for the determination of hemoglobin in unaltered whole blood.
A second object is to provide a method for the determination of hemoglobin in unaltered whole blood, which may be performed in a disposable microcuvette.
A third object is to provide a cuvette with capillary inlet and without active reagents and hemolysing agent for the determination of hemoglobin in unaltered whole blood.
A fourth object is to provide a method of processing results of absorption measurements for determination of hemoglobin in unaltered whole blood.
A fifth object is to provide a system for implementing the methods for the determination of hemoglobin in unaltered whole blood.
Other objects will be apparent from the following description and the accompanying claims.
In accordance with an aspect of the present invention a method for providing such a hemoglobin determination comprises the steps of
providing a disposable, capillary cuvette, which has an optical path length of less than 1 mm;
filling said cuvette with a sample of unaltered whole blood;
performing a first absorption measurement at a wavelength in the range 490-520 nm directly on the sample in the cuvette,
further conducting a second absorption measurement, and
processing results of the first and second absorption measurements to determine the concentration of hemoglobin in the sample, wherein the step of processing comprises compensating for scattering in the sample, said compensating being dependent on the result of the second absorption measurement.
According to another aspect of the present invention a method is provided for determining a concentration of hemoglobin in a sample of undiluted, unhemolyzed whole blood from a result of a first absorption measurement on the sample performed at a wavelength in the range 490-520 nm and a result of a second absorption measurement on the sample. The method comprises: processing the results of the first and second absorption measurements to determine the concentration of hemoglobin in the sample, wherein the step of processing comprises compensating for scattering in the sample, said compensating being dependent on the result of the second absorption measurement.
According to a further aspect of the present invention a system providing such a hemoglobin determination comprises:
means for emitting light at a first wavelength in a first range of 490-520 nm and at a second wavelength in a second range,
a cuvette holder arranged to receive a capillary cuvette, which has an optical path length of less than 1 mm and holds a sample of unaltered whole blood,
a detector for detecting light transmitted through the sample in a first absorption measurement for light in said first range and in a second absorption measurement for light in said second range, and
a processing unit for processing results of the first and second absorption measurements to determine the concentration of hemoglobin in the sample, wherein the processing comprises compensating for scattering in the sample, said compensating being dependent on the result of the second absorption measurement.
It has thus unexpectedly been found that quantitative determinations of hemoglobin can easily be performed without not only the chemical reagents sodium azide and sodium nitrite but also without a hemolysing agent directly on the unaltered, i.e. undiluted and unhemolysed, whole blood. Since the unaltered whole blood contains blood cells, there is substantial scattering of the light in the sample. Thus, it has heretofore been expected that a quantitative hemoglobin determination in undiluted, unhemolyzed whole blood would require detecting and analysing the scattered light. According to the invention, hemoglobin determination may be performed by two absorption measurements without the need for quantitatively knowing the scattering coefficients of the contents of the blood or physically reducing the measured effects of scattered light. It has unexpectedly been found that by compensating for the level of absorption of the sample in the second absorption measurement, the effect of scattering may easily be accounted for. Thus, according to the invention, hemoglobin determination is simple, requiring only two absorption measurements.
In accordance with the present invention it has thus been found that the hygroscopic reagents can be eliminated. Furthermore, it has been found that the time for obtaining the analytical determination may be reduced. As the analyses are performed in large amounts in e.g. hospitals and blood banks, the time aspect is important.
In the context of this application, the term xe2x80x9cabsorption measurementxe2x80x9d should be construed as a measurement related to the absorption in a sample. In an absorption measurement, the intensity of light detected after interacting with a sample is compared with the intensity of light irradiated on the sample. The detected light corresponds to the transmittance through the sample. The light that does not reach the detector is considered to be absorbed. Thus, in the results of the measurements the transmittance may be used instead of the absorption. As the transmittance is the inverse of the absorption, detecting transmittance would still be an absorption measurement. However, the measured absorption does not only correspond to light that has been truly absorbed in the sample, since some of the light has been scattered in the sample so that it does not reach the detector.
Further, the term xe2x80x9cdeterminationxe2x80x9d should be construed as the measurement not necessarily obtaining an absolutely exact value of the concentration of hemoglobin in the sample. Thus, the concentration of hemoglobin is xe2x80x9cdeterminedxe2x80x9d within reasonable margins of error such that the result not merely gives an order of magnitude of the concentration, while not necessarily giving an absolute value.